Chemoradiotherapy (CRT) is a frontline non-surgical treatment for patients with locally advanced head and neck squamous cell carcinoma (HNSCC). Prior work from this group and others demonstrated that epidermal growth factor receptor (EGFR) expression was associated with CRT resistance, and that adding the anti-EGFR antibody cetuximab (CET) to RT improved outcomes in a subset of HNSCC patients. Furthermore, human papilloma virus (HPV) was identified as a strong prognostic marker in HNSCC patients treated with CRT. Nevertheless, a substantial percentage of patients experience CRT resistance with local relapse or distant metastases. There are cun-ently no validated markers to identify which HNSCC patients are most likely to benefit from CRT regimens, and the mechanisms underlying resistance to these regimens, and how to overcome it, remain unclear. These needs are particularly critical for patients with HPV-negative disease. We hypothesize that by systematic gene expression and proteomic profiling of HNSCC tumors from patients treated with CRT regimens, candidate predictive markers can be identified and subsequently validated using tumor annotated specimens from two large randomized phase III trials, Furthermore, we believe that therapeutic strategies for overcoming this resistance can be identified. Our preliminary data supports these hypotheses. Thus far we have identified several novel markers associated with CRT response including Ku80, a double-strand break repair protein; a post-operative RT (PORT) signature; and an epithelial-to-mesenchymal transition (EMT) signature. We have also identified several targets, including telomerase and Chk2, for overcoming radioresistance. Therefore, to address the unmet needs we proposed the following aims: 1) We will develop candidate gene expression and proteomic markers of CRT resistance using archival HNSCC specimens, and will then prioritize them together with our predefined candidates and markers from Projects 1 and 3, for further testing. 2) The top priority markers will be tested and potentially validated using specimens from two large phase III CRT studies (RTOG 0129 & 0522). 3) We will evaluate Which pathways can be targeted to overcome therapeutic resistance of HNSCC cells in vitno. Therefore, this project has the potential to yield validated markers of as well as new strategies for overcoming CRT resistance. The project also benefits from, and contributes to, the efforts of Projects 1 and 3 and other major grant programs investigating related issues including the HN and Lung SPOREs.